Crypts harbor the stem and transit amplifying cell compartments of intestinal epithelium. However, little is known of crypt cellular physiology due to inaccessibility in vivo and past failures of primary culture. Understanding regulation of intracellular pH (pHi) is particularly critical because it affects cell proliferation/ apoptosis an, therefore, processes of tissue regeneration and the potential for malignant transformation. The genetic disease cystic fibrosis (CF) caused by mutations of the HCO3- exporter CFTR exemplifies this concept by demonstrating an alkaline pHi, crypt hyperplasia and a six-fold increase in gastrointestinal neoplasia incidence. We hypothesize that Cftr normally sets the upper limit of crypt cell alkalinity which provides a negative control over the proliferation process. Crypt proliferation is maintained by extracellular Wnt signaling that canonically regulates the transcriptional cofactor of proliferation, -catenin, and other processes of epithelial morphogenesis. Little is known about how pHi affects Wnt signaling in crypts, although studies in non-mammalian systems indicate that an alkaline pHi increases membrane localization of the Wnt transducer Disheveled (Dvl) and thereby its interactions with Wnt receptors. We hypothesize that crypt hyperplasia in CF results from an alkaline pHi which increases Dvl transduction of Wnt/-catenin signaling and other processes of cell migration/epithelial morphogenesis. In normal intestine, rapid crypt proliferation makes crypt progenitor cells susceptible to off-target effects of chemotherapy/radiation, resulting in crypt damage and the symptoms associated with intestinal mucositis. We hypothesize that pharmacological control of pHi in crypt epithelium can be used to indirectly manipulate cell proliferation to minimize intestinal mucositis. Breakthroughs in developmental biology provide a model of regenerating crypt organoids ('enteroids') in 3D gel culture that we have adapted for studies of intestine from wild-type (WT) and Cftr knockout (KO) mice. Extensive preliminary studies have validated the in vivo fidelity of enteroids and developed techniques that provide remarkable access for imaging, electrophysiology and biochemistry of the gel cultures. In Specific Aim 1, acid-base transporters involved in pHi regulation in transit amplifying cells and stem cells of the crypt will be identified and evaluated for pHi regulation in WT and Cftr KO intestine. Specific Aim 2 will test the hypothesis that cell alkalinity in the Cftr KO crypt increaes membrane association of Dvl thereby facilitating canonical Wnt/-catenin signaling for proliferation and noncanonical pathways for increased cell migration and epithelial morphogenesis. Specific Aim 3 will explore pharmacological manipulation of Cftr and acid-base transporters to slow crypt proliferation during the damage phase and increase crypt proliferation during the repair phase of chemotherapy/radiation. The overall objective is to elucidate the pHi physiology of Wnt signaling in crypt progenitor cells, thereby advancing knowledge of pre-neoplastic conditions and introducing a novel therapeutic strategy for controlling gastrointestinal mucositis after chemotherapy/radiation insult.